Abstract 3975: Clonal emergence of resistance to methotrexate in osteosarcoma barcoded PDX cells
Authors: Giuseppe Longo, Sylvester Jusu, Zhongting Zhang, Zhaohui Xu, Wendong Zhang, Michael Roth, Rui Yang, Douglas Harrison, Jonathan Gill, Richard Gorlick
Journal: Cancer Research (2022)

Abstract Introduction: Osteosarcoma is known to have significant intratumoral heterogeneity. In order to elucidate the clonal emergence of resistance to targeted and cytotoxic chemotherapy in osteosarcoma we have worked on barcoding our cell lines derived from patient derived xenograft (PDX) models. Methods: The cell lines were derived from the PDX models by disaggregation and serial culture using STR to confirm the identity of each of the derived cell lines. The cell lines were barcoded using a lentiviral vector to transduce cells with a unique DNA transcript that was incorporated into the tumor cell genome. Two different cytoxicity assays, the Alamar Blu Cytotoxicity test in combination with the Incucyte® Cytotxicity Assay protocol, were used to evaluate the resistance of the PDX cells to the drug Methotrexate (MTX) that is largely used in the treatment of this disease. Further evaluation of the clonality of the resistant cells will be performed by sequencing the barcodes which will be complemented by functional assays of MTX transport, metabolism and target inhibition. Results: The PDX derived cell lines (n=10) were exposed to serial concentrations of MTX with different exposure durations obtaining the IC50 (range 0.23-376 uM) and IC95 (range 0.8- 4500 uM) for each PDX cell line. Subsequently every PDX cell line previously tested was exposed to the IC95 of MTX, in the same conditions used before, in order to obtain clones resistant to MTX. Numerous clones (72 clones from 10 cell lines) have been obtained. For each clone DNA and RNA has been extracted in order to perform sequencing of the DNA barcoded to determine if the genomic signature of the resistant clones is present in a subpopulation at the onset of treatment and if resistance arises from Darwinian selection or from stochastic alterations during and potentially related to treatment lead to emergence of a new resistant phenotype. RNA sequencing (RNA-Seq) will suggest the pathway alterations that may be associated with the emergence of resistance. Functional assays of MTX resistance will further define the basis of the resistance that emerged. Conclusion: Barcoded PDX derived osteosarcoma cell lines have been created with resistant clones obtained after MTX treatment. Sequencing of barcodes, RNA-Seq profiling and functional assays will yield critical information on the clonality and mechanisms of resistance which will be presented. This work was supported by SAA, Inc., The Foster Foundation and the Barbara Epstein Foundation. Richard Gorlick receives support as the H. Grant Taylor, M.D., W.W. Sutow, M.D. and Margaret P. Sullivan, M.D. Distinguished Chair. Citation Format: Giuseppe Longo, Sylvester Jusu, Zhongting Zhang, Zhaohui Xu, Wendong Zhang, Michael Roth, Rui Yang, Douglas Harrison, Jonathan Gill, Richard Gorlick. Clonal emergence of resistance to methotrexate in osteosarcoma barcoded PDX cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3975.